Uncoupler resistance in E. coli Tuv and Cuv is due to the exclusion of uncoupler by the outer membrane.

The uncoupler resistant bacterial strains E. coli Tuv and Cuv share the high deoxycholate sensitivity of the parent strain, Doc S. However, both Tuv and Cuv show greater resistance than Doc S to other detergents. Measurement of the periplasmic volume indicates that the outer membrane of Doc S is freely permeable to both TPP+ and hydroxymethylinulin. Tuv and Cuv are able to exclude these compounds. EDTA treatment was necessary prior to measuring membrane potential in Tuv and Cuv. Under conditions where delta phi could be measured, uncouplers acted to dissipate delta phi with equal potency in all strains. Uncoupler resistant proline uptake in Tuv and Cuv was abolished by EDTA treatment. Transduction experiments with phage P1 showed that uncoupler resistance could be transferred from Tuv to Doc S. Such transductants were no longer sensitive to novabiocin. The gene for uncoupler resistance cotransduced with the gene pyrE (82 min). Plating efficiency experiments with P1 suggests that detergent sensitivity in Doc S arises from an rfa (81 min) mutation. This mutation is no longer present in Tuv.

The expression of the Na+/glucose cotransporter (SGLT1) gene in lamb small intestine during postnatal development.

We have shown previously that the activity and abundance of the intestinal Na+/glucose cotransporter (SGLT1) declines dramatically during the postnatal development of lambs, and that it can be restored in the intestine of ruminant sheep by intra-luminal infusion of D-glucose. The work presented in this paper has followed the expression of the SGLT1 gene along the vertical and horizontal axes of the ovine small intestine during early development, using quantitative in situ hybridisation histochemistry. Along the vertical axis, SGLT1 mRNA was first detectable just below the crypt-villus junction and rose rapidly to a peak level approx. 150 microns above this point. After reaching a maximum, the amount of message gradually declined towards the villus tip. This pattern of mRNA accumulation along the crypt-villus axis was similar in all intestinal positions and age groups. Along the length of the small intestine (horizontal axis), a decline in the level of SGLT1 mRNA was observed first in the distal intestine. This decrease in SGLT1 mRNA was significant in the intestine (75% of length) of 5-week-old lambs when compared to tissue taken from 25 and 50% of length (P < 0.01 and P < 0.02, respectively). However, the observed fall in the expression of this gene during weaning did not coincide with the fall in activity and amount of SGLT1. In adult animals, where the activity of SGLT1 is very low, the amount of message was greatly reduced. This work supports the finding that the expression of SGLT1 is primarily controlled at the post-transcriptional level during the postnatal development of ovine intestine.

Nucleoside uptake by Trichomonas vaginalis.

A rapid sampling technique was used to follow nucleoside uptake by Trichomonas vaginalis. The results indicated that nucleoside uptake is biphasic with time. Adenosine, guanosine, and uridine uptake is carrier mediated, transported substrate is rapidly metabolised to nucleotides. Two separate carriers appear to exist, one which transports all nucleosides and a second which transports adenosine, guanosine and uridine. Both carriers have more than one binding site for nucleosides. The first carrier has sites for adenosine and pyrimidine nucleosides, and a separate site for purine nucleosides. The second carrier has a site for adenosine and uridine and a separate site for guanosine. Adenosine uptake could not be completely inhibited by nitrobenzylthionucleosides. The rate of nucleoside uptake by T. vaginalis is sufficient to sustain growth.

A procedure for electro-elution of DNA from agarose gels.

A simple device is described for efficient and reproducible electro-elution of DNA resolved in agarose gels. DNA (greater than 1 micrograms) is recovered with consistent yields of over 70% into preset elution volumes of 100 to 500 microliters.

Co-expression of a precursor and the mature protein of wheat ribulose-1,5-bisphosphate carboxylase small subunit from a single gene in Escherichia coli.

The cDNA encoding a precursor of wheat ribulose-1,5-bisphosphate carboxylase/oxygenase was inserted in-phase with prokaryotic expression elements in four different vectors. Five expression vectors encoding the small subunit precursors were cloned in Escherichia coli. None of these constructs expressed detectable amounts of the precursor protein, but all directed synthesis of the mature small subunit. The expression of the small subunit was a consequence of an independent, intragenic Shine-Dalgarno sequence optimally located upstream from an ATG specifying the first codon of the mature small subunit portion in the precursor transcript. Similar internal translation signals have been identified in the nuclear-encoded cDNAs of the small-subunit precursors of numerous higher plant genes. The 5' end of the wheat small-subunit precursor was linked with a consensus E. coli DNA sequence such that the modified gene encoded a partial hybrid precursor carrying four additional residues at its amino terminus. The resultant construct, pEI-W3, directed abundant synthesis of both the partially hybrid small-subunit precursor and the mature small subunit, constituting as much as 10% of the total bacterial protein. The bacterially synthesized small subunit precursor was purified to homogeneity. The authenticity of the recombinant protein was verified by its size, immunological properties, amino-terminal sequence, and amino acid composition.

The uncoupling of respiratory-chain phosphorylation by 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole.

1. 30mmum-4,5,6,7-Tetrachloro-2-trifluoromethylbenzimidazole (TTFB) uncouples respiratory-chain phosphorylation. The respiration rate of uncoupled mitochondria is significantly greater than the state 3 rate. Neither added orthophosphate nor ATP is required to sustain this rate. The oligomycin- and octylguanidine-induced inhibitions of respiration are relieved by uncoupling concentrations of TTFB. 2. Uncoupling concentrations of TTFB elicit a high adenosinetriphosphatase activity and inhibit the ATP-dependent succinate-linked reduction of NAD catalysed by submitochondrial particles from ox-heart mitochondria. 3. The action of TTFB is similar to that of 2,4-dinitrophenol. Evidence is presented which shows that it is the anionic forms of benzimidazoles and imidazoles, having the negative charge localized on the ring nitrogen atom, which are the effective uncoupling agents.

The effects of carbodiimides on functions associated with the energy-conservation mechanism in beef heart sub-mitochondrial particles.

N,N'-di-n-propyl-, N,N'di-n-butyl-, N,N'-di-n-pentyl-, N,N'di-n-hexyl-, N,N'di-n-octoyl, N,N'-dibenzhydryl-, and N,N'-dibenzhydrylcarbodiimides were synthesized. They were all effective inhibitors (2 nmoles carbodiimide per milligram protein) of the ATP-driven reduction of NAD by succinate and the ATP-driven transhydrogenase activities catalyzed by beef heart submitochondrial particles (SMP). They had no effect on the nonenergy-linked transhydrogenase and stimulated the succinate-driven aerobic transhydrogenase activity of beef heart SMP. It was concluded that they exert their effects by reacting with the N,N'-dicyclohexylcarbodiimide-binding protein. Water-soluble carbodiimides were not effective inhibitors.

Involvement of the endogenous inhibitor protein in the MgATP-induced inhibition of soluble mitochondrial adenosine triphosphatase activity.

Chloroform-released ATPase from ox heart mitochondria contains significant amounts of inhibitor protein. There is a correlation between processes that affect the interactions between the inhibitor protein and the ATPase molecule and the ability of MgATP to induce an inhibition of ATPase activity. Evidence is presented suggesting that the endogenous inhibitor protein is involved in the process of MgATP-induced inhibition of soluble ATPase activity.

MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase.

1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.

MgATP-induced inhibition of the adenosine triphosphatase activity of submitochondrial particles.

1. The ATP-hydrolytic activity of ox heart submitochondrial particles can be increased from 2-3 mumol/min per mg of protein to 10-12 mumol/min per mg of protein by incubation in media containing 50 mM-Na2B4O7. This process appears to be due to the partial release of inhibitor protein from the particles. 2. The ATPase activity of submitochondrial particles can be inhibited by incubation with the substrate, MgATP. This inhibition is not due to the accumulation of the hydrolysis products, MgADP and Pi, but could involve the process of ATP hydrolysis. 3. The mechanism of MgATP-induced inhibition of ATPase activity is proposed to involve a conformational change in one of the intermediate enzyme species of the ATP-hydrolytic sequence. 4. MgATP inhibits the ATPase activity of control submitochondrial particles at a higher rate and to a greater extent than it does that of inhibitor-protein-depleted submitochondrial particles, suggesting that the conformational change involves the endogenous inhibitor protein.

Evidence for the existence of a channel in the glucose-specific carrier EIIGlc of the Salmonella typhimurium phosphoenolpyruvate-dependent phosphotransferase system.

The effect of membrane-impermeable sulfhydryl reagents on glucose-specific enzyme II (EIIGlc) activity has been studied in Salmonella typhimurium whole cells and in properly sealed inverted cytoplasmic membrane vesicles. Glutathione N-hexylmaleimide and N-polymethylenecarboxymaleimides inactivate methyl alpha-D-glucopyranoside (alpha-MeGlc) transport and phosphorylation in whole cell preparations at a dithiol that can be protected by oxidizing reagents, trivalent arsenicals, or phosphorylation of EIIGlc. Accessibility to this activity-linked site is restricted to small apolar reagents or to polar reagents with a hydrophobic spacer between the polar group and the reactive maleimide moiety. These same reagents inactivate alpha-MeGlc phosphorylation in inverted cytoplasmic membrane vesicles. Inhibition results from reaction at a dithiol that can be protected by nonpermeant mercurials, oxidants, and arsenicals as well as by phosphorylation of EII. The characteristics of this site are virtually identical with those of the activity-linked dithiol elucidated in intact cells. No evidence could be found for a second activity-linked site on the other side of the membrane when the permeable reagent N-ethylmaleimide was used. Since only one activity-linked dithiol can be detected with sealed inverted membrane vesicles or intact cells and it is accessible to membrane-impermeable sulfhydryl reagents from both sides of the cytoplasmic membrane, we suggest that it is located in a channel constructured by the carrier and that the channel spans the membrane. A second dithiol, not essential for activity, is located near the outer surface of the cytoplasmic membrane.

Mechanisms involved in the uptake of D-glucose into the milk producing cells of rat mammary tissue.

There is a rapid stimulation of the rate of efflux of 3-O-methyl D-glucose from intact lactating rat mammary tissue slices in response to a change in the Na(+)-gradient across the basolateral plasma membrane. RNA extracted from lactating mammary glands contains a 4 kb transcript which hybridises with the cDNA for rabbit intestinal SGLT1. A fraction of the mammary gland cells which is enriched in endoplasmic, Golgi and plasma membranes contains a protein that is immunologically similar to the Na(+)-dependent D-glucose symporter present in the plasma membrane of polarised epithelial cells, e.g., rabbit enterocytes, ovine enterocytes and parotid acinar cells.